All non-sensory pertussis toxin sensitive G protein alpha subunits have been knocked out in earlier years by gene targeting. Previously we had found that Gi2 KO mice develop ulcerative colitis (UC) in 129Sv mice but not C57BL mice, that Go KO mice develop a turning syndrome. In collaborative studies we also noted that expression of Go-alpha blocks cAMP-induced neurite outgrowth. [unreadable] [unreadable] During the previous year we completed studies on the cerebellar foliation defect in Gi2 KO mice. We discovered that Gi3 KO mice have a developmental defect in ribcage formation and, in collaboration with Bernd Nuernberg from the University of Duessledorf, Germany, found that Gi3 KO mice fail to show insulins inhibition of liver autophagy. A vector has been made to generate transgenic mice that express Go-alpha only in dopaminergic neurons to test the hypothesis that Go-deficient mice circle because of lack of Go in their dopaminergic neurons. HIT and RUN (also IN and OUT) vectors were designed and genetically engineered mice have been obtained with loxP sites flanking either Gi2 alpha exons 2-3-4, Go exon exon 6, and GNAS exons XXL The response of macrophages to Toll-like receptor agonists has been chracterized.[unreadable] [unreadable] During the present year we completed our program to flox G protein alpha subunits by completing the construction of a vector to flox GNAS exon 1, we obtained mice heterozygous for loss of Gs-alpha, we began new studies on the molecular mechanism by which receptors activate heterotrimeric G proteins, we began the development of a method to survey the genome-wide methylation status of the mammalian genome, we finalized the construction of a transgene to monitor allelic expression of Gs-alpha whereby one allele is marked with green and the other with red fluorescent protein, and have constructed a vector to delete by homologous recombination one of two multiple repeats present in the GNAS locus that may affect its imprinting.[unreadable] [unreadable] The purpose of the transgene - 120 kb in size, made up of DNA derived from three human BACs and spaning from a few kb upstream of the syntaxin 16 gene to a few hundred bases after exon 1 of GNAS, followed by a mini intron and the coding sequence of the fluorescent protein - is to monitor the relative expression defined by the parent of origin of the Gs-alpha transcript - read out as GFP or RFP. The expectation is that this will allow for the first time to assess variations in imprinting at a cell to cell level in mice that are wt or heterozygous for the loss of Gs, and to correlate this variation with changes in phenotype that depend on the parent of origin of the active Gs gene. The transgene will now be injected into pronuclei of fertilize eggs to generate founder mice. One set of green founders and one set of red founders, so as to begin the research aspect of this project. [unreadable] [unreadable] The strategy to assess chages in genome methylation is based on a genomic SAGE approach whereby genomic DNA is cut with the methylation sensitive enzyme HpaII, the unmethylated ends are ligated to PCR adaptors with a recognition site for MmeI, the ligated adaptors are freed by digestion with MmeI, which adds to the ends of the adaptors a 17nt tag that identifies the HpaII site to whch the adaptors were ligated, the tagged adaptors are ligated onto themselves to generate ditags. The mammalian genome has about 20 million CpGs of which 1.5-1.6 are in the context of the CCGG tetrauncleotide sequence. HpaII will therefore inform in a yes-no fashion on the methylation status of 7% of the genomes CpGs. Once obtained, the tagged HpaI sites are then amplified by PCR, the PCR adaptor sequence is cut off, the core ditags are concatamerized, capped with a new set of PCR adaptors that are biotinylated yielding a library of ditags that will be sequenced by the 454 LIfescience technology. The expectation is that through analysis of the unmethylated CpGs from mice having had different environmental exposures it will be possible to define patterns that can be relate to the exposure. Under environmental exposure one should understand sex, age, insults such as inflammation and immunologic stress, defined diseases. A first hypothesis to be tested is whether some-most-none of these patterns can be detected in white blood cells and whether the bone marrow hematopoietic stem cell niche can be used as a reporter niche of life experience. Additional information to be gathered will contribute to the description of differentially methylated domains or regions (DMRs) and whether methylation status can inform about allelic exclusion of which imprinting is but one of many forms in which allelic exclusion is manifested. [unreadable] [unreadable] Of particular interest to the Transmembrane Signaling Group is the mechanism by which allelic exclusion comes about for the transcription unit responsible for generating the mRNA encoding the alpha subunit of the Gs G protein. Depending on the context, we refer to this unit as the GNAS1 or Gs-alpha gene, transcription unit or mRNA. The human clinical syndromes of pseudohypoparathyroidism 1a and 1b (PHP1a and PHP1b, and pseudopseudohypoparathyroidism (PPHP) arise in individuals that are heterozygous for loss of Gs-alpha due to mutations in the structural GNAS1 gene (PHP1a and PPHP), or are heterozygous for loss of Gs-alpha function because of monoallelic exclusion without a mutation in the coding sequence of Gs-alpha - an epigenetic phenomenon. PHP1a is the first and best studied of these syndromes and manifests in individuals that have inherited an inactivating mutation in the coding sequence of Gs-alpha from their mothers. They are +/- in all tissues except a few in which the gene is imprinted and only the maternal allele is expressed. In these tissues the heterozygote is null. The most prominent effect is loss of responsiveness of the renal proximal tubule to parathyroid hormone - a phenomenon discovered at the NIH in the late 1960s - which gave the name to the syndrome. The loss of responsiveness is due to loss of the Gs needed to transduce the PTH-receptor interaction into activation of adenylyl cylcase. We have set up an allele specific RT-PCR assay based on a BanI polymorphism in mice that allows us to measure the relative expression of maternal and paternal alleles if the parents differ in their BanI polymorphism. [unreadable] [unreadable] To our suprise, we have been unable to document imprinting of GNAS1 in mice. Not only in the proximal renal tubule, but also in white and brown fat, the pituitary and the thyroid, all tissues that according to the literature should show imprinting. This held true for 129SV, C57Bl6/J and CD-1 outbred mice. Closer examination of the literature uncovered a publication reporting the same finding for imprinting of Gs-alpha in the renal cortex of human fetal kidney. We are baffled by this finding, for which we have no explanation. By homologous recombination we have now generated a knockout mouse for which the mutation is kept alive by heterozygous breeding since total loss of Gs is early embryionic lethal. By targetted breeding we are introducing a BanI polymorphism into this strain and are setting up to test the imprinting status of heteozygotes to test the hypothesis that the human syndromes some how depend on a structural mutation in one of the alleles in the parent and to test for epigenetic effects we are not aware off. We also are focusing on an 8-fold direct repeat in the GNAS locus for which we are setting up to create a targeted deletion in mouse.[unreadable] [unreadable] Other ongoing studies focus on properties of a Gs-alpha mutant that may inform on the molecular mechanism by which receptors activate Gs. [unreadable] [unreadable] We are collaborating with extramural scientists in the analysis of the phenotypes that arise in G protein alpha KO mice. Most interesting has recently been the failure of eosinophils to extravasate through Gi2-/- endothelia.